Use of erbb3 inhibitors in the treatment of triple negative and basal-like breast cancers

ABSTRACT

Provided are methods of suppressing growth of triple negative breast tumors and basal-like breast tumors by contacting tumor cells with an ErbB3 inhibitor, e.g., an anti-ErbB3 antibody. Also provided are methods for treating triple negative breast cancer or basal-like breast cancer in a patient by administering to the patient an ErbB3 inhibitor, e.g., an anti-ErbB3 antibody. The treatment methods can further comprise selecting a patient having a triple negative breast cancer or basal-like breast cancer and then administering an ErbB3 inhibitor to the patient. The treatment methods also can further comprise administering at least one additional anti-cancer agent to the patient in combination with the ErbB3 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/312895, filed Mar. 11, 2010, entitled, “Use of ErbB3 Inhibitorsin the Treatment of Triple Negative and Basal-Like Breast Cancers” whichis hereby incorporated by reference in its entirety.

BACKGROUND

In women, breast cancer is among the most common cancers and is thefifth most common cause of cancer deaths. Due to the heterogeneity ofbreast cancers, 10-year progression free survival can vary widely withstage and type, from 98% to 10%. Different forms of breast cancers canhave remarkably different biological characteristics and clinicalbehavior. Thus, classification of a patient's breast cancer has become acritical component for determining a treatment regimen. For example,along with classification of histological type and grade, breast cancersnow are routinely evaluated for expression of hormone receptors(estrogen receptor (ER) and progesterone receptor (PR)) and forexpression of HER2 (ErbB2), since a number of treatment modalities arecurrently available that target hormone receptors or the HER2 receptor.ER and PR are both nuclear receptors (they are predominantly located atcell nuclei, although they can also be found at the cell membrane) andsmall molecular inhibitors that target ER and/or PR have been developed.HER2, or human epidermal growth factor receptor type 2, is a receptornormally located on the cell surface and antibodies that target HER2have been developed as therapeutics. HER2 is the only member of the EGFRfamily (which also includes HER1 (EGFR), HER3 (ErbB3) and HER4 (ErbB4)that is not capable of binding to an activating ligand on its own. ThusHER2 is only functional as a receptor when incorporated into aheterodimeric receptor complex with another EGFR family member, such asHER3. Cancers classified as expressing the estrogen receptor (estrogenreceptor positive, or ER⁺ tumors) may be treated with an ER antagonistsuch as tamoxifen. Similarly, breast cancers classified as expressinghigh levels the HER2 receptor may be treated with an anti-HER2 antibody,such as trastuzumab, or with a HER2-active receptor tyrosine kinaseinhibitor such as lapatinib.

Triple negative (TN) breast cancer is a term used to designate awell-defined clinically relevant subtype of breast carcinomas thataccount for approximately 15% of all breast cancer cases. TN tumorsscore negative (i.e., using conventional histopathology methods andcriteria) for expression of ER and PR and do not express amplifiedlevels of HER2 (i.e., they are ER⁻, PR⁻, HER2⁻). TN breast cancercomprises primarily, but not exclusively, a molecularly andhistopathologically distinct subtype of breast cancer known as thebasal-like (BL) subtype. The BL subtype also is characterized by theexpression of cytokeratins (e.g., CK, CK5/6, CK14, CK17) and otherproteins found in normal basal/myoepithelial cells of the breast.However, in addition to the BL subtype, certain other types of breastcancers, including some “normal breast-like”, metaplastic carcinomas,medullary carcinomas and salivary gland-like tumors can also exhibit thetriple negative (TN) phenotype. Furthermore, TN breast cancers occurmore frequently in the presence of BRCA1 mutations and in pre-menopausalfemales of African-American or Hispanic descent. TN tumors typicallydisplay very aggressive behavior, with shorter post-relapse survival andpoor overall survival rates relative to other breast cancer types.

Not all BL breast cancers are TN. Basal-like breast tumors are aheterogeneous tumor type that account for up to 15% of all breastcancers and exhibit aggressive clinical behavior that makes themparticularly difficult to treat successfully. A majority of BL breastcancers are ER-, PR-, and HER2 low (HER2¹⁺ or HER2 negative). Inaddition, they typically express proteins usually found in normal breastbasal (myoepithelial) cells. These include high molecular weightcytokeratins (e.g., 5/6, 8, 14, 17 and 18), p-cadherin, caveolins 1 and2, nestin, αβ crystalline, and EGFR. Furthermore, BL tumor cellstypically lack the capacity for competent homologous recombination DNArepair.

Histologically, most BL breast cancers are of IDC-NST type, highhistological grade, and exhibit very high mitotic indices. They alsotypically have central necrotic or fibrotic zones, pushing borders,conspicuous lymphocytic infiltrates, and typical/atypical medullaryfeatures, and generally exhibit features similar to those of humanpapilloma virus-induced squamous cell carcinoma of the head and neck.

A great majority of medullary and atypical medullary, metaplastic,secretory, myoepithelial, and adenoid cystic carcinomas of the breastalso exhibit BL characteristics.

Given the lack of expression of hormone receptors or of significantamounts of HER2 in TN breast cancer cells, treatment options have beenvery limited as the tumors are not responsive to treatments that targetER (e.g., tamoxifen, aromatase inhibitors) or HER2 (e.g., trastuzumab).Instead these tumors are treated with conventional neoadjuvant andadjuvant chemotherapy regimens, which have limited efficacy and manycytotoxic side effects. Furthermore, such chemotherapy regimens can leadto drug resistance in tumors, and the risk of recurrence of disease inTN breast cancers is higher within the first three years of treatmentthan for other types of breast cancers.

Basal-like breast cancers are also difficult to treat and are associatedwith poor prognoses, though BL adenoid cystic carcinomas generally areassociated with better clinical outcomes . . . use thereof for themanufacture

In view of the foregoing, a need remains for additional treatmentoptions and methods for treating triple negative breast cancers and BLbreast cancers.

SUMMARY

Provided herein are methods for treating triple negative breast cancers(e.g., tumors) and basal-like breast cancers (e.g., tumors), as well aspharmaceutical compositions that can be used in such methods. Themethods and compositions are based, at least in part, on the discoverythat ErbB3 inhibition can suppress the growth of TN breast cancer cellsand BL breast cancer cells. In particular, administration of anti-ErbB3antibody is demonstrated to suppress the growth of TN breast cancercells in vivo.

Accordingly, use of an ErbB3 inhibitor (e.g., use thereof for themanufacture of a medicament) for the treatment of TN or BL breast canceris provided. In another aspect, a method of suppressing growth of a TNbreast cancer tumor or a BL breast cancer tumor is provided, the methodcomprising contacting the tumor with an effective amount of an ErbB3inhibitor. In another aspect, a method of suppressing growth of a TNbreast cancer tumor or BL breast cancer tumor in a patient is provided,the method comprising administering to the patient an effective amountof an ErbB3 inhibitor. In yet another aspect, a method of treating apatient for a TN breast cancer tumor or BL breast cancer tumor isprovided, the method comprising administering to the patient aneffective amount of an ErbB3 inhibitor. In still another aspect, amethod of treating a breast cancer tumor or BL breast cancer tumor in apatient is provided, the method comprising: selecting a patient with atriple negative breast cancer tumor or a BL breast cancer tumor; andadministering to the patient an effective amount of an ErbB3 inhibitor.

In an exemplary embodiment, the ErbB3 inhibitor is an anti-ErbB3antibody. An exemplary anti-ErbB3 antibody is MM-121 (Ab #6), comprisingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 2, respectively.Another exemplary anti-ErbB3 antibody is an antibody comprising,optionally in amino terminal to carboxy terminal order, V_(H) CDR1, 2and 3 sequences as shown in SEQ ID NOs: 3-5, respectively, and,optionally in amino terminal to carboxy terminal order, V_(L) CDR1, 2and 3 sequences as shown in SEQ ID NOs: 6-8, respectively. In otherembodiments, the anti-ErbB3 antibody is Ab #3 (comprising V_(H) andV_(L) sequences as shown in SEQ ID NOs: 9 and 10, respectively), Ab #14(comprising V_(H) and V_(L) sequences as shown in SEQ ID NOs: 17 and 18,respectively), Ab #17 (comprising V_(H) and V_(L) sequences as shown inSEQ ID NOs: 25 and 26, respectively) or Ab #19 (comprising V_(H) andV_(L) sequences as shown in SEQ ID NOs: 33 and 34, respectively). Instill other embodiments, the anti-ErbB3 antibody is selected from thegroup consisting of mAb 1B4C3, mAb 2D1D12, AMG-888 and humanized mAb8B8. In another embodiment, administration of the anti-ErbB3 antibodyinhibits growth or invasiveness or metastasis of the tumor.

The methods provided herein can be used in the treatment of TN breastcancers of various different histopathological phenotypes. For example,in one embodiment, the triple negative breast cancer tumor ishistopathologically characterized as having a basal-like phenotype. Inanother embodiment, the TN breast cancer tumor is histopathologicallycharacterized as having a phenotype other than BL.

In each of the foregoing methods and compositions, the ErbB3 inhibitormay be comprised in a formulation comprising a pharmaceuticallyacceptable carrier.

In another aspect, the treatment methods provided herein furthercomprise administering to the patient at least one additionalanti-cancer agent that is not an ErbB3 inhibitor. In one embodiment, theat least one additional anti-cancer agent comprises at least onechemotherapeutic drug, such as a drug(s) selected from the groupconsisting of platinum-based chemotherapy drugs, taxanes, tyrosinekinase inhibitors, and combinations thereof. It has now been observedthat in the subset of TN breast cancers that test HER2²⁺, treatment withanti-HER2 agents such as trastuzumab, pertuzumab or lapatinib mayprovide benefits when used in combination with anti-ErbB3 antibodies.Thus in another aspect the treatment methods provided herein furthercomprise administering to the patient an effective amount of at leastone additional anti-cancer agent that is an anti-HER2 agent. Suchanti-HER2 agents are well known and may include one or more ofanti-ErbB2 antibodies such as C6.5 (and the numerous derivativesthereof) described in U.S. Pat. No. 5,977,322, trastuzumab, as describedin U.S. Pat. No. 6,054,297, and pertuzumab, as described in U.S. Pat.No. 6,949,245; as well as small molecule anti-HER2 agents such aslapatinib (which also inhibits EGFR tyrosine kinase) and AG879.

In another embodiment, the at least one additional anti-cancer agentcomprises an EGFR inhibitor, such as an anti-EGFR antibody or a smallmolecule inhibitor of EGFR signaling. An exemplary anti-EGFR antibodycomprises cetuximab. Other examples of anti-EGFR antibodies includematuzumab, panitumumab, nimotuzumab and mAb 806. An exemplary smallmolecule inhibitor of EGFR signaling comprises gefitinib. Other examplesof useful small molecule inhibitors of EGFR signaling include lapatinib,canertinib, erlotinib HCL, pelitinib, PKI-166, PD158780, and AG 1478.

In yet another embodiment, the at least one additional anti-cancer agentcomprises a VEGF inhibitor. An exemplary VEGF inhibitor comprises ananti-VEGF antibody, such as the bevacizumab antibody.

In another embodiment, administration of the anti-ErbB3 antibody and theat least one additional anti-cancer agent inhibits growth orinvasiveness or metastasis of the tumor.

In another aspect, methods of treating TN breast cancer or BL breastcancer in a patient comprise administering to said patient a combinationcomprising MM-121 and paclitaxel. In one embodiment the combinationexhibits therapeutic synergy in the treatment of TN or BL breastcancers. In some examples, the combination effects a log₁₀ cell kill ofat least 2.8, at least 2.9 or at least 3.0. In other aspects, thecombination provides an improvement in tumor growth inhibition that isat least about additive as compared to improvement obtained with each ofthe single agents of the combination.

In another embodiment, there is provided a composition comprising acombination of MM-121 and paclitaxel, wherein the combination exhibitstherapeutic synergy in the treatment of TN or BL breast cancers. In someexamples, the composition effects a log₁₀ cell kill of at least 2.8, atleast 2.9 or at least 3.0.

Kits containing the combination pharmaceutical compositions also areprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing relative MAXF449 xenograft tumor volume (%) (Yaxis—normalized to initial tumor volume) plotted against time in daysfollowing randomization (X axis) in NMRI nude mice treated with MM-121or vehicle control. TGI=200%.

FIG. 2 is a graph showing the percent change in MDA-MB-231 xenografttumor volume (Y axis—normalized to initial tumor volume) plotted againsttime in days following injection of MDA-MB-231 cells (X axis) in Balb/cnude mice treated with MM-121 or vehicle control. Curves with opentimepoint squares or circles indicate mice treated with MM-121. Curveswith filled timepoint squares or circles indicate vehicle controls. Inthe inset, “mp” indicates that the MDA-MB-231 cells were injected intothe mammary fat pad, while “sc” indicates that the MDA-MB-231 cells wereinjected subcutaneously in the flank.

FIG. 3 is a graph showing MDA-MB-231 tumor volume in mm³ (Y axis)plotted against time in days (X-axis) starting at 28 days followinginjection of MDA-MB-231 cells into the mammary fat pads of Balb/c nudemice. Treatment was with MM-121 (150 μg/mouse), paclitaxel (5 mg/kg), acombination of MM-121 (150 μg/mouse) and paclitaxel (5 mg/kg), orvehicle control. Where used in the figures, “mpk”=mg/kg.

FIG. 4 presents graphs showing MDA-MB-231 tumor volume in mm³ (Y axis)plotted against time in days (X-axis) starting at 28 days followinginjection of MDA-MB-231 cells into the mammary fat pads of Balb/c nudemice. FIG. 4A depicts treatment with MM-121, cetuximab, or paclitaxel;MM-121 and cetuximab; and the triple combination MM-121 and cetuximaband paclitaxel. FIG. 4B depicts treatment with MM-121, erlotinib, MM-121and erlotinib, or the triple combination of MM-121 and erlotinib andpaclitaxel.

DETAILED DESCRIPTION

Provided herein are methods for treating triple negative and basal-likebreast cancers, as well as pharmaceutical compositions that can be usedin practicing such methods. As described further in the Examples, it hasnow been demonstrated that an ErbB3 inhibitor, in particular ananti-ErbB3 antibody, is able to suppress the growth of TN breast cancercells in vivo. Accordingly, methods for suppressing the growth of TNbreast cancers and BL breast cancers, as well as methods of treatingsuch breast cancers in patients, using an ErbB3 inhibitor are providedherein.

Definitions:

As used herein, the term “triple negative” or “TN” refers to tumors(e.g., carcinomas), typically breast tumors, in which the tumor cellsscore negative (i.e., using conventional histopathology methods) forestrogen receptor (ER) and progesterone receptor (PR), both of which arenuclear receptors (i.e., they are predominantly located at cell nuclei),and the tumor cells are not amplified for epidermal growth factorreceptor type 2 (HER2 or ErbB2), a receptor normally located on the cellsurface. Tumor cells are considered negative for expression of ER and PRif less than 5% of the tumor cell nuclei are stained for ER and PRexpression using standard immunohistochemical techniques. Tumor cellsare considered highly amplified for HER2 (“HER2³⁺”) if, when tested witha HercepTest™ Kit (Code K5204, Dako North America, Inc., Carpinteria,Calif.), a semi-quantitative immunohistochemical assay using apolyclonal anti-HER2 primary antibody, they yield a test result score of3+, or, the test HER2 positive by fluorescence in-situ hybridization(FISH). As used herein, tumor cells are considered negative for HER2overexpression if they yield a test result score of 0 or 1+, or 2+, orif they are HER2 FISH negative.

Furthermore, the term “triple negative breast cancer(s)” or “TN breastcancer(s)” encompasses carcinomas of differing histopathologicalphenotypes. For example, certain TN breast cancers are classified as“basal-like” (“BL”), in which the neoplastic cells express genes usuallyfound in normal basal/myoepithelial cells of the breast, such as highmolecular weight basal cytokeratins (CK, CK5/6, CK14, CK17), vimentin,p-cadherin, αβ crystallin, fascin and caveolins 1 and 2. Certain otherTN breast cancers, however, have a different histopathologicalphenotype, examples of which include high grade invasive ductalcarcinoma of no special type, metaplastic carcinomas, medullarycarcinomas and salivary gland-like tumors of the breast.

The terms “ErbB3,” “HER3,” “ErbB3 receptor,” and “HER3 receptor,” asused interchangeably herein, refer to human ErbB3 protein, as describedin U.S. Pat. No. 5,480,968.

As used herein, the term “ErbB3 inhibitor” is intended to includetherapeutic agents that inhibit, downmodulate, suppress or downregulateactivity of ErbB3. The term is intended to include chemical compounds,such as small molecule inhibitors, and biologic agents, such asantibodies, interfering RNA (shRNA, siRNA), soluble receptors and thelike. An exemplary ErbB3 inhibitor is an anti-ErbB3 antibody.

An “antibody,” as used herein is a protein consisting of one or morepolypeptides comprising binding domains substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes, wherein theprotein immunospecifically binds to an antigen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively. A typical immunoglobulin structural unitcomprises a tetramer that is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). “V_(L)” and V_(H)″ refer to these lightand heavy chains respectively.

Antibodies include intact immunoglobulins as well as antigen-bindingfragments thereof, which may be produced by digestion with variouspeptidases, or synthesized de novo either chemically or usingrecombinant DNA expression technology. Such fragments include, forexample, F(ab)₂ dimers and Fab monomers. Useful antibodies includesingle chain antibodies (antibodies that exist as a single polypeptidechain), e.g., single chain Fv antibodies (scFv) in which a V_(H) and aV_(L) chain are joined together (directly or through a peptide linker)to form a continuous polypeptide.

“Immunospecific” or “immunospecifically” refer to antibodies that bindvia domains substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes to one or more epitopes of a protein ofinterest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicmolecules. Typically, an antibody binds immunospecifically to a cognateantigen with a K_(d) with a value of no greater than 50 nM, as measuredby a surface plasmon resonance assay or a cell binding assay. The use ofsuch assays is well known in the art, and is described in Example 3,below.

An “anti-ErbB3 antibody” is an antibody that immunospecifically binds tothe ectodomain of ErbB3 and an “anti-ErbB2 antibody” is an antibody thatimmunospecifically binds to the ectodomain of ErbB2. The antibody may bean isolated antibody. Such binding to ErbB3 or ErB2 exhibits a K_(d)with a value of no greater than 50 nM as measured by a surface plasmonresonance assay or a cell binding assay. An anti-ErbB3 antibody may bean isolated antibody. Exemplary anti-ErbB3 antibodies inhibit EGF-likeligand mediated phosphorylation of ErbB3. EGF-like ligands include EGF,TGFa, betacellulin, heparin-binding epidermal growth factor, biregulin,epigen, epiregulin, and amphiregulin, which typically bind to ErbB1 andinduce heterodimerization of ErbB1 with ErbB3.

As used herein, the term “EGFR inhibitor” is intended to includetherapeutic agents that inhibit, downmodulate, suppress or downregulateEGFR signaling activity. The term is intended to include chemicalcompounds, such as small molecule inhibitors (e.g., small moleculetyrosine kinase inhibitors) and biologic agents, such as antibodies,interfering RNA (shRNA, siRNA), soluble receptors and the like.

As used herein, the term “VEGF inhibitor” is intended to includetherapeutic agents that inhibit, downmodulate, suppress or downregulateVEGF signaling activity. The term is intended to include chemicalcompounds, such as small molecule inhibitors (e.g., small moleculetyrosine kinase inhibitors) and biologic agents, such as antibodies,interfering RNA (shRNA, siRNA), soluble receptors and the like.

The terms “suppress”, “suppression”, “inhibit” and “inhibition” as usedinterchangeably herein, refer to any statistically significant decreasein biological activity (e.g., tumor cell growth), including fullblocking of the activity. For example, “inhibition” can refer to adecrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%in biological activity.

The term “patient” includes a human or other mammalian animal thatreceives either prophylactic or therapeutic treatment.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures such as those described herein. Themethods of “treatment” employ administration to a patient of an ErbB3inhibitor provided herein, for example, a patient having a TN or BLbreast cancer tumor, in order to prevent, cure, delay, reduce theseverity of, or ameliorate one or more symptoms of the disease ordisorder or recurring disease or disorder, or in order to prolong thesurvival of a patient beyond that expected in the absence of suchtreatment.

The term “effective amount,” as used herein, refers to that amount of anagent, such as an ErbB3 inhibitor, for example an anti-ErbB3 antibody,which is sufficient to effect treatment, prognosis or diagnosis of a TNor BL breast cancer, when administered to a patient. A therapeuticallyeffective amount will vary depending upon the patient and diseasecondition being treated, the weight and age of the patient, the severityof the disease condition, the manner of administration and the like,which can readily be determined by one of ordinary skill in the art. Thedosages for administration can range from, for example, about 1 ng toabout 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg,about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng toabout 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg,about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg, about 30μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50 μg toabout 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg to about2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175 mg,about 500 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg toabout 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg,about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mgto about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg,about 500 mg, or about 525 mg to about 625 mg, of an antibody or antigenbinding portion thereof, as provided herein. Dosing may be, e.g., everyweek, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks orevery 6 weeks. Dosage regimens may be adjusted to provide the optimumtherapeutic response. An effective amount is also one in which any toxicor detrimental effects (side effects) of the agent are minimized and/oroutweighed by the beneficial effects. For MM-121, administration may beintravenous at exactly or about 6 mg/kg or 12 mg/kg weekly, or 12 mg/kgor 24 mg/kg biweekly. Additional dosing regimens are described below.

The terms “anti-cancer agent” and “antineoplastic agent” refer to drugsused to treat malignancies, such as cancerous growths. Drug therapy maybe used alone, or in combination with other treatments such as surgeryor radiation therapy.

Various aspects and embodiments are described in further detail in thefollowing subsections.

I. ErbB3 Inhibitors

As described in further detail herein, the methods and compositionsprovided herein involve the use of one or more ErbB3 inhibitors.

In one embodiment, the ErbB3 inhibitor is an anti-ErbB3 antibody, e.g.,a monoclonal antibody. An exemplary anti-ErbB3 monoclonal antibody isMM-121, described further in WO 2008/100624 and U.S. Pat. No. 7,846,440,and having V_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 2,respectively. Alternately, the anti-ErbB3 monoclonal antibody is anantibody that competes with MM-121 for binding to ErbB3. In anotherembodiment, the anti-ErbB3 antibody is an antibody comprising the V_(H)and V_(L) CDR sequences of MM-121, which are shown in SEQ ID NOs: 3-5(V_(H) CDR1, 2, 3) and 6-8 (V_(L) CDR1, 2, 3), respectively. Otherexamples of anti-ErbB3 antibodies include Ab #3, Ab #14, Ab #17 and Ab#19, also described further in WO 2008/100624 and having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 9 and 10, 17 and 18, 25 and 26, and 33and 34 respectively. In another embodiment, the anti-ErbB3 antibody isan antibody comprising the V_(H) and V_(L) CDR sequences of Ab # 3(shown in SEQ ID NOs: 11-13 and 14-18, respectively) or antibodycomprising the V_(H) and V_(L) CDR sequences of Ab # 14 (shown in SEQ IDNOs: 19-21 and 22-24, respectively) or an antibody comprising the V_(H)and V_(L) CDR sequences of Ab # 17 (shown in SEQ ID NOs: 27-29 and30-32, respectively) or an antibody comprising the V_(H) and V_(L) CDRsequences of Ab # 19 (shown in SEQ ID NOs: 35-37 and 38-40,respectively).

Alternately, the anti-ErbB3 antibody is a monoclonal antibody or antigenbinding portion thereof which binds an epitope of human ErbB3 comprisingresidues 92-104 of SEQ ID NO:41 and is characterized by inhibition ofproliferation of a cancer cell expressing ErbB3. The cancer cell may bea MALME-3M cell, an AdrR cell, or an ACHN cell and the proliferation maybe reduced by at least 10% relative to control. In an additionalembodiment this isolated monoclonal antibody or antigen binding portionthereof binds an epitope comprising residues 92-104 and 129 of SEQ IDNO:41.

Other examples of useful anti-ErbB3 antibodies include the antibodies1B4C3 and 2D1D12 (U3 Pharma AG), both of which are described in USPatent Application Publication No. 20040197332 by Ullrich et al., andmonoclonal antibodies (including humanized versions thereof), such asAMG-888 (U3 Pharma AG and Amgen) and 8B8, as described in U.S. Pat. No.5,968,511 by Akita et al.

In yet another embodiment, the anti-ErbB3 antibody can comprise amixture, or cocktail, of two or more anti-ErbB3 antibodies, each ofwhich binds to a different epitope on ErbB3. In one embodiment, themixture, or cocktail, comprises three anti-ErbB3 antibodies, each ofwhich binds to a different epitope on ErbB3.

In another embodiment, the ErbB3 inhibitor comprises a nucleic acidmolecule, such as an RNA molecule, that inhibits the expression oractivity of ErbB3. RNA antagonists of ErbB3 have been described in theart (see e.g., US Patent Application Publication No. 20080318894).Moreover, interfering RNAs specific for ErbB3, such as shRNAs or siRNAsthat specifically inhibits the expression and/or activity of ErbB3, havebeen described in the art.

In yet another embodiment, the ErbB3 inhibitor comprises a soluble formof the ErbB3 receptor that inhibits signaling through the ErbB3 pathway.Such soluble ErbB3 molecules have been described in the art (see e.g.,U.S. Pat. No. No. 7,390,632, U.S. Patent Application Publication No.20080274504 and U.S. Patent Application Publication No. 20080261270,each by Maihle et al., and U.S. Patent Application Publication No.20080057064 by Zhou).

II. Methods

In one aspect, use of an ErbB3 inhibitor for the manufacture of amedicament for the treatment of TN breast cancer or BL breast cancer isprovided.

In another aspect, a method of suppressing growth of a triple negativebreast cancer cell is provided, the method comprising contacting thecell with an effective amount of an ErbB3 inhibitor.

In another aspect, a method of suppressing growth of a TN or BL breastcancer tumor in a patient is provided, the method comprisingadministering to the patient an effective amount of an ErbB3 inhibitor.

In yet another aspect, a method of treating a patient for a TNor BLbreast cancer tumor is provided, the method comprising administering tothe patient an effective amount of an ErbB3 inhibitor.

In still another aspect, a method of treating a breast cancer tumor in apatient is provided, the method comprising:

selecting a patient with a TN or BL breast cancer tumor; and

administering to the patient an effective amount of an ErbB3 inhibitor.

In another aspect, the patient with a TN or BL breast cancer tumor is apatient further selected by use of the selection methods disclosed inpending international application PCT/US2009/054051.

Identification of a triple negative breast cancer cells, or a patienthaving a triple negative breast cancer tumor, can be achieved throughstandard methods well known in the art. For example, immunohistochemical(IHC) staining is routinely used in biopsy analysis and permits thedetection, localization and relative quantification of ER, PR, and HER2within sections from formalin-fixed, paraffin-embedded tissues (e.g.,breast cancer tissues routinely processed for histological evaluation).In the context of identifying TN tumors, staining of less than 5% oftumor cell nuclei is considered negative for each of for ER and PR. Theprimary antibody used for IHC staining of ER is e.g., 1D5 (Chemicon,Temecula Calif., catalog # IHC2055). The primary antibody used for IHCstaining of PR is e.g., PgR636 (Thermo Fisher Scientific, Fremont,Calif., catalog # MS-1882-R7) or PgR 1294 (Dako North America, Inc.,Carpinteria, Calif., Code M3568). The ErbB2 IHC assay used is e.g., theHercepTest™ Kit (Dako North America, Inc., Carpinteria, Calif., CodeK5204), a semi-quantitative IHC assay using a polyclonal anti-HER2primary antibody to determine HER2 protein overexpression in breastcancer tissues routinely processed for histological evaluation, which isused according to the manufacturer's directions. In the context ofidentifying TN tumors, a test result of 0 to 1+ is considered Her2negative.

In one embodiment, the triple negative breast cancer tumor ishistopathologically characterized as having a basal-like phenotype. Inanother embodiment, the triple negative breast cancer tumor ishistopathologically characterized as having a phenotype other thanbasal-like. Examples of TN breast cancer histopathological phenotypesthat are other than BL include high grade invasive ductal carcinoma ofno special type, metaplastic carcinomas, medullary carcinomas andsalivary gland-like tumors of the breast.

In one aspect, the TN or BL breast cancer to be treated with ErbB3inhibitor coexpresses ErbB 1 (EGFR), ErbB3, and heregulin (HRG).Expression of EGFR and HRG can be identified by RT-PCR or by standardimmunoassay techniques, such as ELISA assay or immunohistochemicalstaining of formalin-fixed, paraffin-embedded tissues (e.g., breastcancer tissues routinely processed for histological evaluation), usingan anti-EGFR antibody, anti-ErbB3 antibody or an anti-HRG antibody.Additional characteristics of tumors for treatment in accordance withthe disclosure herein are set forth in pending U.S. Patent PublicationNo. 20110027291, which claims priority to PCT application No.PCT/US2009/054051.

In one embodiment, the ErbB3 inhibitor administered to the patient is ananti-ErbB3 antibody. An exemplary anti-ErbB3 antibody is MM-121,comprising V_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 2,respectively, or an antibody comprising V_(H) CDR1, 2 and 3 sequences asshown in SEQ ID NOs: 3-5, respectively, and V_(L) CDR1, 2 and 3sequences as shown in SEQ ID NOs: 6-8, respectively (i.e., the V_(H) andV_(L) CDRs of MM-121). Additional non-limiting exemplary anti-ErbB3antibodies and other forms of ErbB3 inhibitors are described in detailin Subsection I above.

The ErbB3 inhibitor can be administered to the patient by any routesuitable for the effective delivery of the inhibitor to the patient. Forexample, many small molecule inhibitors are suitable for oraladministration. Antibodies and other biologic agents typically areadministered parenterally, e.g., intravenously, intraperitoneally,subcutaneously or intramuscularly. Various routes of administration,dosages and pharmaceutical formulations suitable for use in the methodsprovided herein are described in further detail below.

III. Pharmaceutical Compositions

In another aspect, pharmaceutical compositions are provided that can beused in the methods disclosed herein, i.e., pharmaceutical compositionsfor treating TN or BL breast cancer tumors.

In one embodiment, the pharmaceutical composition for treating TN breastcancer comprises an ErbB3 inhibitor and a pharmaceutical carrier. TheErbB3 inhibitor can be formulated with the pharmaceutical carrier into apharmaceutical composition. Additionally, the pharmaceutical compositioncan include, for example, instructions for use of the composition forthe treatment of patients for TN or BL breast cancer tumors.

In one embodiment, the ErbB3 inhibitor in the composition is ananti-ErbB3 antibody, e.g., MM-121 or an antibody comprising the V_(H)and V_(L) CDRs of MM-121 positioned in the antibody in the same relativeorder as they are present in MM-121 so as to provide immunospecificbinding of ErbB3. Additional non-limiting exemplary anti-ErbB3antibodies and other forms of ErbB3 inhibitors are described in detailin Subsection I above.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, and otherexcipients that are physiologically compatible. Preferably, the carrieris suitable for parenteral, oral, or topical administration. Dependingon the route of administration, the active compound, e.g., smallmolecule or biologic agent, may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion, as well as conventionalexcipients for the preparation of tablets, pills, capsules and the like.The use of such media and agents for the formulation of pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the pharmaceutical compositions provided herein iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutically acceptable carrier can include a pharmaceuticallyacceptable antioxidant. Examples of pharmaceutically-acceptableantioxidants include: (1) water soluble antioxidants, such as ascorbicacid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,sodium sulfite and the like; (2) oil-soluble antioxidants, such asascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and (3) metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions provided herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof, andinjectable organic esters, such as ethyl oleate. When required, properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants. In manycases, it will be useful to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, monostearate salts and gelatin.

These compositions may also contain functional excipients such aspreservatives, wetting agents, emulsifying agents and dispersing agents.

Therapeutic compositions typically must be sterile, non-phylogenic, andstable under the conditions of manufacture and storage. The compositioncan be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization, e.g., by microfiltration. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation include vacuum drying and freeze-drying (lyophilization)that yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theactive agent(s) may be mixed under sterile conditions with additionalpharmaceutically acceptable carrier(s), and with any preservatives,buffers, or propellants which may be required.

Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Pharmaceutical compositions comprising an ErbB3 inhibitor can beadministered alone or in combination therapy. For example, thecombination therapy can include a composition provided herein comprisingan ErbB3 inhibitor and at least one or more additional therapeuticagents, such as one or more chemotherapeutic agents known in the art,discussed in further detail in Subsection IV below. Pharmaceuticalcompositions can also be administered in conjunction with radiationtherapy and/or surgery.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

Exemplary dosage ranges for administration of an antibody include:10-1000 mg (antibody)/kg (body weight of the patient), 10-800 mg/kg,10-600 mg/kg, 10-400 mg/kg, 10-200 mg/kg, 30-1000 mg/kg, 30-800 mg/kg,30-600 mg/kg, 30-400 mg/kg, 30-200 mg/kg, 50-1000 mg/kg, 50-800 mg/kg,50-600 mg/kg, 50-400 mg/kg, 50-200 mg/kg, 100-1000 mg/kg, 100-900 mg/kg,100-800 mg/kg, 100-700 mg/kg, 100-600 mg/kg, 100-500 mg/kg, 100-400mg/kg, 100-300 mg/kg and 100-200 mg/kg. Exemplary dosage schedulesinclude once every three days, once every five days, once every sevendays (i.e., once a week), once every 10 days, once every 14 days (i.e.,once every two weeks), once every 21 days (i.e., once every threeweeks), once every 28 days (i.e., once every four weeks) and once amonth.

It may be advantageous to formulate parenteral compositions in unitdosage form for ease of administration and uniformity of dosage. Unitdosage form as used herein refers to physically discrete units suited asunitary dosages for the patients to be treated; each unit contains apredetermined quantity of active agent calculated to produce the desiredtherapeutic effect in association with any required pharmaceuticalcarrier. The specification for unit dosage forms are dictated by anddirectly dependent on (a) the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions disclosed herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. “Parenteral” as usedherein in the context of administration means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

The phrases “parenteral administration” and “administered parenterally”as used herein refer to modes of administration other than enteral(i.e., via the digestive tract) and topical administration, usually byinjection or infusion, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion. Intravenous injectionand infusion are often (but not exclusively) used for antibodyadministration.

When agents provided herein are administered as pharmaceuticals, tohumans or animals, they can be given alone or as a pharmaceuticalcomposition containing, for example, 0.001 to 90% (e.g., 0.005 to 70%,e.g., 0.01 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

IV. Combination Therapy

In certain embodiments, the methods and uses provided herein forsuppressing growth of TN breast cancer cells or for treating a patientwith a TN breast tumor or BL breast tumor can comprise administration ofan ErbB3 inhibitor and at least one additional anti-cancer agent that isnot an ErbB3 inhibitor.

In one embodiment, the at least one additional anti-cancer agentcomprises at least one chemotherapeutic drug. Non-limiting examples ofsuch chemotherapeutic drugs include platinum-based chemotherapy drugs(e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel (Taxol®),docetaxel (Taxotere®), EndoTAG-1™ (a formulation of paclitaxelencapsulated in positively charged lipid-based complexes; MediGene),Abraxane® (a formulation of paclitaxel bound to albumin)), tyrosinekinase inhibitors (e.g., imatinib/Gleevec®, sunitinib/Sutent®,dasatinib/Sprycel®), and combinations thereof.

In another embodiment, the at least one additional anti-cancer agentcomprises an EGFR inhibitor, such as an anti-EGFR antibody or a smallmolecule inhibitor of EGFR signaling. An exemplary anti-EGFR antibody iscetuximab (Erbitux®). Cetuximab is commercially available from ImCloneSystems Incorporated. Other examples of anti-EGFR antibodies includematuzumab (EMD72000), panitumumab (Vectibix®; Amgen); nimotuzumab(TheraCIM™) and mAb 806. An exemplary small molecule inhibitor of theEGFR signaling pathway is gefitinib (Iressa®), which is commerciallyavailable from AstraZeneca and Teva. Other examples of small moleculeinhibitors of the EGFR signaling pathway include erlotinib HCL (OSI-774;Tarceva®, OSI Pharma); lapatinib (Tykerb®, GlaxoSmithKline); canertinib(canertinib dihydrochloride, Pfizer); pelitinib (Pfizer); PKI-166(Novartis); PD158780; and AG 1478(4-(3-Chloroanillino)-6,7-dimethoxyquinazoline).

In yet another embodiment, the at least one additional anti-cancer agentcomprises a VEGF inhibitor. An exemplary VEGF inhibitor comprises ananti-VEGF antibody, such as bevacizumab (Avastatin®; Genentech).

In still another embodiment, the at least one additional anti-canceragent comprises an anti-ErbB2 antibody. Suitable anti-ErbB2 antibodiesinclude trastuzumab and pertuzumab.

In one aspect, the improved effectiveness of a combination according tothe invention can be demonstrated by achieving therapeutic synergy.

The term “therapeutic synergy” is used when the combination of twoproducts at given doses is more efficacious than the best of each of thetwo products alone at the same doses. In one example, therapeuticsynergy can be evaluated by comparing a combination to the best singleagent using estimates obtained from a two-way analysis of variance withrepeated measurements (e.g., time factor) on parameter tumor volume.

The term “additive” refers to when the combination of two or moreproducts at given doses is equally efficacious than the sum of theefficacies obtained with of each of the two or more products, whilst theterm “superadditive” refers to when the combination is more efficaciousthan the sum of the efficacies obtained with of each of the two or moreproducts.

Another measure by which effectiveness (including effectiveness ofcombinations) can be quantified is by calculating the log₁₀ cell kill,which is determined according to the following equation:

log₁₀cell kill=T−C(days)/3.32×T _(d)

in which T−C represents the delay in growth of the cells, which is theaverage time, in days, for the tumors of the treated group (T) and thetumors of the control group (C) to have reached a predetermined value (1g, or 10 mL, for example), and T_(d) represents the time, in daysnecessary for the volume of the tumor to double in the control animals.When applying this measure, a product is considered to be active iflog₁₀ cell kill is greater than or equal to 0.7 and a product isconsidered to be very active if log₁₀ cell kill is greater than 2.8.

Using this measure, a combination, used at its own maximum tolerateddose, in which each of the constituents is present at a dose generallyless than or equal to its maximum tolerated dose, exhibits therapeuticsynergy when the log₁₀ cell kill is greater than the value of the log₁₀cell kill of the best constituent when it is administered alone. In anexemplary case, the log₁₀ cell kill of the combination exceeds the valueof the log₁₀ cell kill of the best constituent of the combination by atleast one log cell kill.

EXAMPLES Example 1 MM-121 Effects on triple Negative Human Breast CancerXenograft MAXF449

An analysis of the anti-tumor efficacy and tolerability of MM-121treatment of tumor-bearing mice is carried out using triple negativehuman mammary carcinoma xenograft MAXF449 (ONCOTEST GmbH, Frieburg,Germany) in NMRI nude mice. MAXF449 is a Human tumor explant(histologically described upon explant as solid invasive ductal, andpoorly defined) established via subcutaneous injection in serialpassages in nude mice. The MAXF449 cells used in these experiments havebeen passaged 22 times. NMRI nude mice are obtained from Taconic farms,Charles River Laboratories International, or Harlan Laboratories. Themice are housed in Tecniplast Individually Ventilated polycarbonate(Macrolon) Cages (IVC) set in climate-controlled rooms and have freeaccess to food and acidified water.

To investigate anti-tumor efficacy in monotherapy, MM-121 or vehiclecontrol (100 μL) is given to tumor-bearing mice at 600 μg per mouse(MM-121 as a 6 mg/mL solution in PBS) by IP injection every three days.Control mice receive the PBS vehicle only. Efficacy is determined bycomparing tumor growth between the antibody-treated mice and the vehiclecontrol mice and is expressed as the experimental to control ratio ofmedian relative tumor volumes (T/C value). A minimum T/C value below 50%is a prerequisite for rating a treatment as effective. The control andexperimental groups each contain 10 mice bearing one tumor each. Toobtain 30 mice bearing tumors of similar sizes for randomization, 40mice per tumor are implanted unilaterally.

Mice are randomized and therapy begins when a sufficient number ofindividual tumors have grown to a volume of approximately 200 mm3.Tumors are measured (L×W) by digital caliper measurement and the tumorvolume is calculated using the formula Pi/6 (W2×L). The first dose isadministered either on Day 0 (day of randomization) or one day later.

Approximately 24 hours after administration of the final dose all miceare bled to prepare serum; in addition, tumors are collected from thesame mice for flash-freezing and FFPE (½ tumor each).

According to regulations for animal experiments, mice are sacrificed ifthe tumor volume exceeds 1800 mm³ (one tumor per mouse). Mice aremonitored and dosed until their tumors have grown to that size but nolonger than 60 days. Thereafter, they are sacrificed for samplecollection.

At the end of the study, approximately 24 hours after administration ofthe final dose, all mice on study are bled sublingually to obtain amaximum amount of blood for the preparation of serum. Serum is aliquotedin 2 tubes with approximately 250 μL in each.

In addition, tumors from all mice are excised without delay forsnap-freezing in liquid nitrogen (½ tumor, COVARIS bags for the storageof samples are provided) and for fixation in 10% buffered formalin for<24 hours, subsequent dehydration and paraffin embedding (FFPE, ½tumor).

Animal weights and tumor diameters (W and L) are measured twice weeklyand tumor volumes are calculated using the formula Pi/6 (W2×L). Tumorgrowth curves are plotted. Tumor inhibition and absolute growth delayfor 2 and 4 doubling times are calculated.

Results of experiments that were carried out substantially as describedare presented in FIG. 1. MM-121 treatment inhibited or stopped tumorgrowth, and in some cases reduced tumor size. TGI (tumor growthinhibition) in these human triple negative tumor xenografts wascalculated to be approximately 200%.

Example 2 MM-121 Effects on Triple-Negative Human Breast CancerXenograft MDA-MB-231

Balb/c nude mice are injected under general anesthesia with 10⁷MDA-MB-231 human triple negative breast cancer cells (ATCC) eithersubcutaneously in the flank or into the mammary fat pad. Mice withestablished tumors (i.e., after 7-10 days of tumor growth followinginjection of cells) are then treated IP with either PBS or MM-121 every3 days with 600 ug MM-121 per mouse as described in Example 1. Tumorvolume is measured twice a week as described in Example 1.

Results of experiments carried out substantially as described arepresented in FIG. 2. MM-121 treatment stopped human triple negativebreast cancer tumor growth essentially completely in these experiments.

Example 3 Measurement of Binding Affinity (K_(D))

The dissociation constants of anti-ErbB antibodies may be measured usingeither or both of two independent techniques, a Surface PlasmonResonance Assay and a cell binding assay.

Surface Plasmon Resonance Assay

The Surface Plasmon Resonance Assay is performed as described in Wassafet al. (2006) Analytical Biochem., 351:241-253. One implementation usesa BIACORE 3000 instrument (GE Healthcare) using a recombinant ErbBprotein as the analyte and the anti-ErbB antibody as the ligand TheK_(D) value is calculated based on the formula K_(D)=K_(d)/K_(a).

Cell Binding Assay

A cell binding assay is performed using MALME-3M cells (ATCC) for ErbB3binding. The assay is performed substantially as follows.

Cells are detached with 2 mLs trypsin-EDTA+2 mLs RMPI+5 mM EDTA at roomtemperature for 5 minutes. Complete RPMI (10 mLs) is added immediatelyto the trypsinized cells, resuspended gently and spun down in a Beckmantabletop centrifuge at 1100 rpm for 5 minutes. Cells are resuspended inBD stain buffer (PBS+2% FBS+0.1% sodium azide, Becton Dickinson) at aconcentration of 2×10⁶ cells per ml and 50 μl (1×10⁵ cells) aliquots areplated in a 96-well titer plate.

A 150 μl solution of 200 nM anti-ErbB antibody in BD stain buffer isprepared and serially diluted 2-fold into 75 μl BD stain buffer. Theconcentrations of the diluted antibody ranged from 200 nM to 0.4 nM. 50μl aliquots of the different protein dilutions are then added directlyto the 50 ul cell suspension giving the final concentrations of 100 nM,50 nM, 25 nM, 12 nM, 6 nM, 3 nM, 1.5 nM, 0.8 nM, 0.4 nM and 0.2 nM ofthe antibody.

Aliquoted cells in the 96-well plate are incubated with the proteindilutions for 30 minutes at room temperature on a platform shaker andwashed 3 times with 300 μl BD stain buffer. Cells are then incubatedwith 100 μl of secondary antibody (e.g., a 1:750 dilution of Alexa647-labeled goat anti-human IgG in BD stain buffer) for 45 minutes on aplatform shaker in the cold room. Finally, cells are washed twice,pelleted and resuspended in 250 μl BD stain buffer+0.5 μg/ml propidiumiodide. Analysis of 10,000 cells is done in a FACSCALIBUR flow cytometerusing the FL4 channel. MFI values and the corresponding concentrationsof the anti-ErbB-antibody are plotted on the y-axis and x-axis,respectively. The K_(D) of the molecule is determined using GraphPadPRISM software using the one-site binding model for a non-linearregression curve.

The K_(D) value is calculated based on the formula Y=Bmax*X/K_(D)+X(Bmax=fluorescence at saturation. X=antibody concentration. Y=degree ofbinding).

Example 4 Inhibition of Tumor Growth in Vivo by Combination TreatmentWith MM-121 and Paclitaxel Methods:

Balb/c nude mice (female, 4-5 weeks old from Charles River lab) areimplanted orthotopically with 10×106 cells in mammary pad. Tumors areallowed to reach average of 100 mm³ in size before randomization into 4groups of 10 mice, containing mice with a similar size distribution oftumors. Each group of mice is treated with 1) MM-121 (150 ug/mouse, ip.,Q3D) or 2) vehicle control (PBS, ip.) or 3) paclitaxel (5 mg/kg LC Labs)or 4) paclitaxel (5 mg/kg) and MM-121 (150 ug/mouse). Treatment iscontinued for 4 weeks. Tumors are measured twice weekly and tumor volumeis calculated as p/6×length×width², where the width is the shortermeasurement.

Results:

The combination of MM-121 with paclitaxel was investigated in vivo inthe MDA-MB-231 triple negative breast cancer xenograft model using themethods described above or minor variations thereof. Mice were treatedwith sub-optimal doses of MM-121, paclitaxel, a combination of MM-121and paclitaxel, or vehicle control (FIG. 3). While both MM-121 andpaclitaxel each inhibited tumor growth in vivo, mice receiving acombination therapy of MM-121 and paclitaxel exhibited an improvement oftumor growth inhibition when compared to that obtained with each of theindividual treatments. The improvement in tumor growth inhibitionexhibited therapeutic synergy and was at least about additive ascompared to the improvement obtained with each of the single agents ofthe combination.

Table 1 shows data used to generate FIG. 3. Table 2 shows the mean %change in tumor volumes using data from the same experiments shown inFIG. 3, normalized to initial tumor volume.

TABLE 1 data used to generate FIG. 3 - mean tumor volumes in mm³ VehicleMean 104.4 137.1 144.5 229.5 253.7 291.0 MM121 150 μg Mean 99.4 115.5137.5 180.4 187.2 242.7 paclitaxel 5 mg/kg Mean 97.9 113.5 144.6 166.2178.8 202.2 MM121 150 μg + Mean 96.2 100.8 98.3 104.1 113.0 121.6paclitaxel 5 mg/kg

Example 5 MM-121 Combination With Targeted and Chemotherapies In VivoMethods:

Balb/c nude mice (female, 4-5 weeks old from Charles River lab) areimplanted orthotopically with 10×106 cells in mammary pad. Tumors areallowed to reach average of 150 mm³ in size before randomization into 9groups of 8 mice, containing mice with a similar size distribution oftumors. Each group of mice is treated with a dose of 1)MM-121(300ug/mouse, ip., Q3D) or 2) vehicle control (PBS, ip.) or 3)paclitaxel (10 mg/kg LC Labs) or 4) erlotinib (50 mg/kg PO 5XQD) or 5)cetuximab (2 mg/kg Q3D) or combination therapy with: 6) erlotinib (50mg/kg) and MM-121 (300 ug/mouse), or 7) cetuximab (2 mg/kg) and MM-121(300 ug/mouse), or 8) erlotinib (50 mg/kg) and MM-121 (300 ug/mouse) andpaclitaxel (10 mg/kg), or 9) cetuximab (2 mg/kg) and MM121 (300ug/mouse) and paclitaxel (10 mg/kg). Treatment is continued for 4 weeks.Tumors are measured twice weekly and tumor volume is calculated asp/6×length×width², where the width is the shorter measurement.

Results: In order to test the efficacy of MM-121 to inhibit tumor growthwhen used in combination with other agents, these combinations weretested in vivo in the MDA-MB-231 triple negative breast cancer xenograftmodel using the methods described above or minor variations thereof.Mice were treated with MM-121 (administered at sub-optimal doses in thecombinations), cetuximab, paclitaxel, MM-121 and cetuximab, and thetriple combination MM-121 and cetuximab and paclitaxel. As shown in FIG.4A, combination therapy with MM-121 and cetuximab inhibited tumor growthto a greater extent than either agent alone and essentially stoppedtumor growth until at least day 39. The decreased rate of growth showedtherapeutic synergy and, in certain cases represented at least about anadditive decrease in growth compared to the decreased rates obtainedwith any of the single therapies. Addition of paclitaxel did not enhancethe effect of MM-121 and cetuximab. Mice were then treated with MM-121,erlotinib, MM-121 and erlotinib, or the triple combination of MM-121 anderlotinib and paclitaxel. As shown in FIG. 4B, MM-121 in combinationwith erlotinib did not have a statistically significant effect on therate of tumor growth compared with treatment with either agent alone.Conversely, treatment with the triple combination of MM-121, erlotinib,and paclitaxel resulted in a clearly decreased rate of tumor growth andessentially stopped tumor growth until at least day 39. The decreasedrate of growth showed therapeutic synergy and, in certain casesrepresented at least about an additive decrease in growth compared tothe decreased rates obtained with any of the single or double therapies.

Table 3 shows data used to generate FIGS. 4A and 4B. Table 4 shows themean % change in tumor volume using data from the same experiments shownin FIGS. 4A and 4B, normalized to initial tumor volume.

TABLE 2 Data used to generate FIGS. 4A and 4B - mean tumor volumes inmm³. Day 28 32 36 39 43 46 49 53 PBS 163.7 199.0 242.8 304.5 369.4 423.4458.4 490.7 MM121 300 ug 178.6 197.3 219.1 257.8 269.4 291.4 351.3 425.0erlotinib 172.1 182.1 216.1 273.2 252.8 245.6 303.1 327.4 50 mg/kgcetuximab 172.4 210.6 245.0 269.2 296.3 279.7 283.5 358.1 2 mg/kgMM121 + 170.6 215.5 221.8 261.7 272.5 255.3 305.2 378.3 erlotinibpaclitaxel 155.2 167.0 182.4 216.6 228.1 247.0 292.6 383.5 10 mg/kgMM121 + 152.5 149.6 171.6 169.1 196.6 171.2 182.9 241.2 cetuximabMM121 + 164.8 149.3 139.8 146.5 156.7 163.4 202.9 264.5 erlotinib +paclitaxel MM121 + 176.3 158.5 147.8 160.4 154.4 163.4 203.4 247.7cetuximab + paclitaxel

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims. Any combinations of the embodimentsdisclosed in the dependent claims are contemplated to be within thescope of the invention.

Incorporation by Reference

Each and every, issued patent, patent application and publicationreferred to herein is hereby incorporated herein by reference in itsentirety.

SUMMARY OF SEQUENCE LISTING MM-121 V_(H) amino acid sequence(SEQ ID NO: 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGL KMATIFDYWGQGTLVTVSSMM-121 V_(L) amino acid sequence (SEQ ID NO: 2)QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSSIFV IFGGGTKVTVLMM-121 V_(H) CDR1 (SEQ ID NO: 3) HYVMA MM-121 V_(H) CDR2 (SEQ ID NO: 4)SISSSGGWTLYADSVKG MM-121 V_(H) CDR3 (SEQ ID NO: 5) GLKMATIFDYMM-121 V_(L) CDR1 (SEQ ID NO: 6) TGTSSDVGSYNVVS MM-121 V_(L) CDR2(SEQ ID NO: 7) EVSQRPS MM-121 V_(L) CDR3 (SEQ ID NO: 8) CSYAGSSIFVI Ab #3 V_(H) amino acid sequence (SEQ ID NO: 9)EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNMRWVRQAPGKGLEWVSVIYPSGGATRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGY YYYGMDVWGQGTLVTVSSAb # 3 V_(L) amino acid sequence (SEQ ID NO: 10)QSVLTQPPSASGTPGQRVTISCSGSDSNIGRNYIYWYQQFPGTAPKLLIYRNNQRPSGVPDRISGSKSGTSASLAISGLRSEDEAEYHCGTWDDSLSGPV FGGGTKLTVL Ab #3 V_(H) CDR1 (SEQ ID NO: 11) AYNMR Ab # 3 V_(H) CDR2 (SEQ ID NO: 12)VIYPSGGATRYADSVKG Ab # 3 V_(H) CDR3 (SEQ ID NO: 13) GYYYYGMDV Ab #3 V_(L) CDR1 (SEQ ID NO: 14) SGSDSNIGRNYIY Ab # 3 V_(L) CDR2(SEQ ID NO: 15) RNNQRPS Ab # 3 V_(L) CDR3 (SEQ ID NO: 16) GTWDDSLSGPVAb # 14 V_(H) amino acid sequence (SEQ ID NO: 17)EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSYISPSGGHTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLETGLLVDAFDIWGQGTMVTVSS Ab # 14 V_(L) amino acid sequence (SEQ ID NO: 18)QYELTQPPSVSVYPGQTASITCSGDQLGSKFVSWYQQRPGQSPVLVMYKDKRRPSEIPERFSGSNSGNTATLTISGTQAIDEADYYCQAWDSSTYVFGTG TKVTVL Ab #14 V_(H) CDR1 (SEQ ID NO: 19) AYGMG Ab # 14 V_(H) CDR2 (SEQ ID NO: 20)YISPSGGHTKYADSVKG Ab # 14 V_(H) CDR3 (SEQ ID NO: 21) VLETGLLVDAFDI Ab #14 V_(L) CDR1 (SEQ ID NO: 22) SGDQLGSKFVS Ab # 14 V_(L) CDR2(SEQ ID NO: 23) YKDKRRPS Ab # 14 V_(L) CDR3 (SEQ ID NO: 24) QAWDSSTYVAb # 17 V_(H) amino acid sequence (SEQ ID NO: 25)EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMGWVRQAPGKGLEWVSYISPSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLN YYYGLDVWGQGTTVTVSSAb # 17 V_(L) amino acid sequence (SEQ ID NO: 26)QDIQMTQSPSSLSASVGDRITITCQASQDIGDSLNWYQQKPGKAPRLLIYDASNLETGVPPRFSGSGSGTDFTFTFRSLQPEDIATYFCQQSANAPFTFG PGTKVDIK Ab #17 V_(H) CDR1 (SEQ ID NO: 27) WYGMG Ab # 17 V_(H) CDR2 (SEQ ID NO: 28)YISPSGGITVYADSVKG Ab # 17 V_(H) CDR3 (SEQ ID NO: 29) LNYYYGLDV Ab #17 V_(L) CDR1 (SEQ ID NO: 30) QASQDIGDSLN Ab # 17 V_(L) CDR2(SEQ ID NO: 31) DASNLET Ab # 17 V_(L) CDR3 (SEQ ID NO: 32) QQSANAPFTAb # 19 V_(H) amino acid sequence (SEQ ID NO: 33)EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMWWVRQAPGKGLEWVSYIGSSGGPTYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGR GTPYYFDSWGQGTLVTVSSAb # 19 V_(L) amino acid sequence (SEQ ID NO: 34)QYELTQPASVSGSPGQSITISCTGTSSDIGRWNIVSWYQQHPGKAPKLMI YDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL Ab # 19 V_(H) CDR1(SEQ ID NO: 35) RYGMW Ab # 19 V_(H) CDR2 (SEQ ID NO: 36)YIGSSGGPTYYVDSVKG Ab # 19 V_(H) CDR3 (SEQ ID NO: 37) GRGTPYYFDS Ab #19 V_(L) CDR1 (SEQ ID NO: 38 TGTSSDIGRWNIVS Ab # 19 V_(L) CDR2(SEQ ID NO: 39) DVSNRPS Ab # 19 V_(L) CDR3 (SEQ ID NO: 40) SSYTSSSTWVErbB3 (SEQ ID NO: 41) SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT

1-53. (canceled)
 54. A method of treating triple negative breast cancer,the method comprising: identifying a patient with a triple negativebreast cancer tumor, and administering to the identified patient aneffective amount of an ErbB3 inhibitor.
 55. The method of claim 54,wherein the administration of the effective amount of the ErbB3inhibitor suppresses growth of the triple negative breast cancer tumor.56. The method of claim 54, wherein the ErbB3 inhibitor comprises ananti-ErbB3 antibody.
 57. The method of claim 56, wherein the anti-ErbB3antibody comprises, in amino terminal to carboxy terminal order, a V_(H)CDR1 sequence as shown in SEQ ID NO:3, a V_(H) CDR2 sequence as shown inSEQ ID NO:4 and a V_(H) CDR3 sequence as shown in SEQ ID NO:5, and, inamino terminal to carboxy terminal order, a V_(L) CDR1 sequence as shownin SEQ ID NO:6, a V_(L) CDR2 sequence as shown in SEQ ID NO:7 and aV_(L) CDR3 sequence as shown in SEQ ID NO:8.
 58. The method of claim 56,wherein the anti-ErbB3 antibody is selected from the group consistingof: (a) an antibody comprising a V_(H) sequence as shown in SEQ ID NO:1and a V_(L) sequence as shown in SEQ ID NO:2; (b) an antibody comprisinga V_(H) sequence as shown in SEQ ID NO:9 and a V_(L) sequence as shownin SEQ ID NO:10; (c) an antibody comprising a V_(H) sequence as shown inSEQ ID NO:17 and a V_(L) sequence as shown in SEQ ID NO:18; and (d) anantibody comprising a V_(H) sequence as shown in SEQ ID NO:25 and aV_(L) sequence as shown in SEQ ID NO:26.
 59. The method of claim 54,wherein identifying the patient includes histopathologicallycharacterizing the breast cancer tumor as having a basal-like phenotype.60. The method of claim 54, wherein identifying the patient includeshistopathologically characterizing the breast cancer tumor as having aphenotype other than basal-like.
 61. The method of claim 54, whichfurther comprises administering to the patient an effective amount of atleast one additional anti-cancer agent.
 62. The method of claim 61,wherein the at least one additional anti-cancer agent is selected fromthe group consisting of platinum-based chemotherapy drugs, taxanes,tyrosine kinase inhibitors, anti-EGFR antibodies, anti-ErbB2 antibodies,and combinations thereof.
 63. The method of claim 62, wherein the atleast one drug is paclitaxel.
 64. The method of claim 61, wherein the atleast one additional anti-cancer agent comprises an EGFR inhibitor. 65.The method of claim 64, wherein the EGFR inhibitor comprises ananti-EGFR antibody.
 66. The method of claim 65, wherein the anti-EGFRantibody is selected from the group consisting of cetuximab, matuzumab,panitumumab, nimotuzumab and mAb
 806. 67. The method of claim 64,wherein the EGFR inhibitor is a small molecule inhibitor of EGFRsignaling selected from the group consisting of gefitinib, lapatinib,canertinib, pelitinib, erlotinib HCL, PKI-166, PD158780, and AG 1478.68. The method of claim 61, wherein the at least one additionalanti-cancer agent comprises a VEGF inhibitor.
 69. The method of claim68, wherein the VEGF inhibitor comprises bevacizumab.
 70. The method ofclaim 54, wherein the triple negative breast cancer tumor is a tumorwhich scores negative for estrogen receptor (ER) and progesteronereceptor and yields a test result of 0, 1+, or 2+ using asemi-quantitative immunohistochemical assay using a polyclonal anti-HER2primary antibody.
 71. The method of claim 70, wherein the tumor is FISHnegative for HER2 gene amplification.
 72. A method of treating triplenegative breast cancer, the method comprising: identifying a patientwith a basal-like breast cancer tumor; and administering to theidentified patient an effective amount of an ErbB3 inhibitor.
 73. Themethod of claim 72, wherein the administration of the effective amountof the ErbB3 inhibitor suppresses growth of the basal-like breast cancertumor.
 74. The method of claim 72, wherein the ErbB3 inhibitor comprisesan anti-ErbB3 antibody that comprises, in amino terminal to carboxyterminal order, a V_(H) CDR1 sequence as shown in SEQ ID NO:3, a V_(H)CDR2 sequence as shown in SEQ ID NO:4 and a V_(H) CDR3 sequence as shownin SEQ ID NO:5, and, in amino terminal to carboxy terminal order, aV_(L) CDR1 sequence as shown in SEQ ID NO:6, a V_(L) CDR2 sequence asshown in SEQ ID NO:7 and a V_(L) CDR3 sequence as shown in SEQ ID NO:8,or wherein the anti-ErbB3 antibody comprises an antibody comprising aV_(H) sequence as shown in SEQ ID NO:1 and a V_(L) sequence as shown inSEQ ID NO:2.
 75. The method of claim 74, which further comprisesadministering to the patient an effective amount of at least oneadditional anti-cancer agent.
 76. The method of claim 75, wherein the atleast one additional anti-cancer agent is paclitaxel.